Systems and methods for minimization or elimination of diffusion effects in a microfluidic system

ABSTRACT

The present invention relates to systems and methods for minimizing or eliminating diffusion effects. Diffused regions of a segmented flow of multiple, miscible fluid species may be vented off to a waste channel, and non-diffused regions of fluid may be preferentially pulled off the channel that contains the segmented flow. Multiple fluid samples that are not contaminated via diffusion may be collected for analysis and measurement in a single channel. The systems and methods for minimizing or eliminating diffusion effects may be used to minimize or eliminate diffusion effects in a microfluidic system for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 12/758,395, filed on Apr. 12, 2010, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/168,395,filed on Apr. 10, 2009, which is incorporated herein by reference in itsentirety.

BACKGROUND

1. Field of the Invention

The present invention relates to systems and methods for minimizingand/or eliminating diffusion effects in a microfluidic system. Morespecifically, embodiments of the present invention relate to systems andmethods for minimizing and/or eliminating diffusion effects in amicrofluidic system having one or more channels so that concentrationdependent measurements can be made on a segmented flow of multiplemiscible fluids in the one or more channels.

2. Description of Related Art

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, correct identification of crime scene features, theability to propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer. One of the mostpowerful and basic technologies to detect small quantities of nucleicacids is to replicate some or all of a nucleic acid sequence many times,and then analyze the amplification products. Polymerase Chain Reaction(“PCR”) is perhaps the most well-known of a number of differentamplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies of the amplified DNA to be detected and analyzed. For generaldetails concerning PCR, see Sambrook and Russell, Molecular Cloning—ALaboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 2005) and PCR Protocols A Guide to Methods andApplications, M. A. Innis et al., eds., Academic Press Inc. San Diego,Calif. (1990).

Once there are a sufficient number of copies of the original DNAmolecule, the DNA can be characterized. One method of characterizing theDNA is to examine the DNA's dissociation behavior as the DNA transitionsfrom double stranded DNA (dsDNA) to single stranded DNA (ssDNA). Theprocess of causing DNA to transition from dsDNA to ssDNA with increasingtemperature is sometimes referred to as a “high-resolution temperature(thermal) melt (HRTm)” process, or simply a “high-resolution melt”process. Alternatively, the transition from ssDNA to dsDNA may beobserved through various electrochemical methods, which generate adynamic current as the potential across the system is changed.

Microfluidic chips are being developed for “lab-on-a-chip” devices toperform in-vitro diagnostic testing. The largest growth area is inmolecular biology where DNA amplification is performed in the sealedchannels of the chip. Optical detection devices are commonly used tomeasure the increasing amplicon product over time (Real Time PCR) and/orto perform a thermal melt to identify the presence of a specificgenotype (High Resolution Thermal Melt). Exemplary disclosures relatedto the imaging of a microfluidic chip to measure the fluorescent productcan be found in commonly-owned U.S. application Ser. No. 11/505,358 toHasson et al. entitled “Real-Time PCR in Micro Channels” (U.S. Pat. Pub.2008-0003588) and U.S. application Ser. No. 11/606,204 to Hasson et al.entitled “Systems and Methods for Monitoring the Amplification andDissociation Behavior of DNA Molecules” (U.S. Pat. Pub. 2008-0003594),the respective disclosures of which are hereby incorporated byreference.

When a fluid is introduced into a channel to measure increasing ampliconproduct in the fluid over time and/or to identify the presence of aspecific genotype in the fluid, it is desirable to minimize and/orprevent contamination of the fluid so that accurate results may beobtained. At the same time, it may be desirable to introduce a series offluid species into a channel so that a single channel may be used tomeasure and/or identify multiple fluid species in succession.Minimization and/or prevention of contamination becomes especiallydifficult when the multiple fluid species are miscible (i.e., capable ofbeing mixed) and are supplied to a single channel in a segmented fashion(i.e., with each species occupying the entire width of the channel andexisting axially down or upstream from another of the species, whichalso occupies the entire width of the channel).

Flow through a microfluidic channel is generally characterized bylaminar flow with parabolic velocity profiles. These parabolic velocityprofiles indicate that fluid along the walls of the microfluidic channelwill move much slower than the fluid at the center of the channel. Inflows of only one species of fluid where the same chemical concentrationexists at all points in the flow, this variation in fluid velocity as afunction of distance from the channel wall has little impact. In flowsin which two miscible species of fluid exist in a segmented fashion, theeffects of the laminar velocity profile are problematic. In particular,as the fluid in the center of the channel moves faster than the edges,each segment of flow will stretch into the segment immediatelydownstream of it. This stretching dramatically increases the surfacearea between each segment of fluid. Due to the different species offluids being miscible in each other, the increase in surface areaincreases the rate of diffusion and therefore the potential crosscontamination between segments. As a result, downstream measurementsthat require significant sections of non-diffused fluids may becomedifficult or impossible to perform due to contamination.

Accordingly, a need exists in the art for systems and methods to ensurethat samples can be obtained in microfluidic systems that are free orsubstantially free from contamination by diffused fluids.

SUMMARY

The present invention relates to systems and methods for minimizingand/or eliminating diffusion effects in a microfluidic system. Thepresent invention allows for concentration dependent measurements to bemade on a segmented flow of multiple miscible fluids in a singlechannel.

In one aspect, the present invention provides a method of collecting,from a continuous flow of two or more miscible fluids sequentiallypresent in a channel, one or more samples that are substantially freefrom contamination by the other miscible fluids present in the channel.In one embodiment, the method comprises: (a) identifying and monitoringthe position of a diffusion region between uncontaminated portions of afirst miscible fluid and a second miscible fluid in a first channel; (b)diverting the diffusion region into a second channel; and (c) collectinga portion of the second miscible fluid which is substantially free fromcontamination by any miscible fluids adjacent to the second misciblefluid. In some embodiments, the monitoring the position of the diffusionregion may include monitoring a leading edge and a trailing edge of thediffusion region.

In still other embodiments, the portion of the second miscible fluidsubstantially free from contamination may be collected in a thirdchannel. The position of the diffusion region may be monitored inrelation to an opening of the third channel. In one embodiment, thediverting may begin at or before a leading edge of the diffusion regionreaches an opening of the third channel and end after a trailing edge ofthe diffusion region passes the opening of the third channel. In otherembodiments, the second channel may lead to a waste area. In otherembodiments, the diverting step may include pulling fluid of thediffusion region from the edge of the first channel, and the collectingstep may include pulling fluid of the portion of the second misciblefluid from the center of the first channel.

In some embodiments, the method may further comprise: (d) identifyingand monitoring the position of a diffusion region between uncontaminatedportions of the second miscible fluid and a third miscible fluid in afirst channel; (e) diverting the diffusion region into the secondchannel; and (f) collecting a portion of the third miscible fluid whichis substantially free from contamination by any miscible fluids adjacentto the third miscible fluid. In other embodiments, the method mayfurther comprise monitoring the amplification of DNA in the collectedportion of the second miscible fluid, and monitoring the dissociationbehavior of the amplified DNA in the collected portion of the secondmiscible fluid.

In other aspects, the present invention provides a microfluidic systemcomprising: a first channel, a second channel, a monitoring device, anda fluid flow control system. In one embodiment, the monitoring deviceidentifies and tracks one or more diffusion regions between adjacentmiscible fluids of two or more miscible fluids present in themicrofluidic system. The fluid flow control system controls the flow offluid through each of the first and second channels. As two or moremiscible fluids are moved through the first channel, the fluid flowcontrol system causes the one or more diffusion regions to flow into anopening of the second channel and causes one or more miscible fluidsamples that are substantially free from contamination by adjacentmiscible fluids present in the first channel to be collected. In someembodiments, the monitoring device may track a leading edge and atrailing edge of the one or more diffusion regions. In otherembodiments, the collected one or more miscible fluid samples may becaused by the fluid flow control system to be collected after a trailingedge of one of the one or more diffusion regions has passed an entranceto a third channel, and the fluid flow control system may causecollection to be stopped before the leading edge of a subsequentdiffusion region reaches the entrance to the third channel.

In some embodiments, the fluid flow control system may control the flowof fluid through each of the first and second channels based on theidentification and tracking of the one or more regions of diffusionperformed by the monitoring device. In other embodiments, the secondchannel may lead to a waste area. The microfluidic system may comprise athird channel in which the one or more miscible fluid samples arecollected. The monitoring device may track a position of the diffusionregion in relation to an opening of the third channel.

In another aspect, the present invention provides a genomic analysissystem comprising: a microfluidic chip, a monitoring device, and a fluidflow control system. In one embodiment, the microfluidic chip has one ormore microfluidic channels and a diffusion effect minimization orelimination system for each of the one or more microfluidic channels.Each of the one or more microfluidic channels passes through a diffusioneffect minimization or elimination zone, a PCR processing zone, and aHRTm analysis zone. The diffusion effect minimization or eliminationsystem comprises a first pathway, second pathway and a third pathway. Asegmented flow of multiple, miscible fluid species enters through thefirst pathway. One or more miscible fluid samples that are substantiallyfree from contamination by adjacent miscible fluids present in the firstpathway are collected and passed to the PCR processing zone and the HRTmanalysis zone through the second pathway. One or more diffusion regionsbetween adjacent fluid species of the segmented flow are diverted to awaste zone through the third pathway. In some embodiments, themonitoring device identifies and tracks the one or more diffusionregions present in the diffusion effect minimization or elimination zoneof the microfluidic chip. In other embodiments, the system furthercomprises a fluid flow control system configured to control the flow offluid through the diffusion effect minimization or elimination systemfor each of the one or more microfluidic channels.

In another aspect, a method of collecting a sample from a continuousflow of two or more miscible fluids sequentially present in a channel isprovided which comprises identifying the leading region of diffusionbetween two sequential fluids, monitoring the passage of the region ofdiffusion through the channel such that once the leading region ofdiffusion has passed an opening for a second channel a portion of thefluid is collected into a second channel, wherein the collecting offluid is stopped before the next sequential region of diffusion reachesthe opening of the second channel. In an embodiment, the collectedsample is free from contamination by the other miscible fluid(s) presentin the channel.

In another aspect, a microfluidic system is provided which comprises afirst channel, a second channel which is in fluid communication with thefirst channel, and means for moving a liquid through each of the firstand second channels. In one embodiment, the microfluidic system furthercomprises means for identifying and tracking region(s) of diffusionbetween two or more miscible fluids present in the first channel,wherein as one or more miscible fluids are moved through the firstchannel, after the region of diffusion has passed the opening of thesecond channel, the means for moving a liquid through the second channelis activated to cause a portion of the fluid to enter the secondchannel, whereby the means for moving a liquid through the secondchannel is deactivated prior to the next region of diffusion passing theopening of the second channel.

In another aspect, the present invention provides a method ofcollecting, from a continuous flow of two or more miscible fluidssequentially present in a first microfluidic channel, one or moresamples that are substantially free from contamination by the othermiscible fluids present in the first channel. The method comprisesflowing the continuous flow of two or more miscible fluids through thefirst channel for a predetermined time. The predetermined time issufficient to allow the diffused region of the at least two misciblefluids to pass a collection region of the first channel. The collectionregion may be a junction of the first channel with a second channel. Themethod also comprises collecting a portion of one of the misciblefluids, which is substantially free from contamination by any misciblefluids adjacent to the collected miscible fluid, into the secondchannel. The method further comprises stopping the collection of one ofthe miscible fluids before a subsequent diffused region reaches thecollection region of the first channel. In an embodiment, thepredetermined time is determined by using a predetermined flow rateand/or a fixed volume displacement. In another embodiment, the steps offlowing, collecting and stopping are repeated for each sample ofmiscible fluid which is substantially free from contamination by theother miscible fluids to be collected. In some embodiments, the firstchannel and the second channel are microfluidic channels.

In another aspect, the present invention provides a system ofcollecting, from a continuous flow of two or more miscible fluidssequentially present in a first channel, one or more samples that aresubstantially free from contamination by the other miscible fluidspresent in the channel. The system comprises the first channel, a secondchannel, a collection region of the first channel formed at a junctionbetween the first channel and the second channel, and a fluid flowcontrol system that controls the flow of fluid through each of the firstand second channels. The fluid flow control system is configured to flowthe continuous flow of the two or more miscible fluids through the firstchannel for a predetermined time. The predetermined time is sufficientto allow the diffused region of the at least two miscible fluids to passthe collection region of the first channel. The fluid flow controlsystem is configured to collect a portion of one of the miscible fluids,which is substantially free from contamination by any miscible fluidsadjacent to the collected miscible fluid, into the second channel. Thefluid flow control system is further configured to stop the collectionof one of the miscible fluids before a subsequent diffused regionreaches the collection region of the channel. In some embodiments, thefirst channel and the second channel are microfluidic channels. In someembodiments, the fluid flow control system is configured to determinethe predetermined time by using a predetermined flow rate and/or a fixedvolume displacement. In some embodiments, the fluid flow control systemis configured to repeat the flowing, collecting and stopping for eachsample of miscible fluid which is substantially free from contaminationby the other miscible fluids to be collected.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIGS. 1A-1D provide schematic diagrams of a diffusion minimizationsystem according to one embodiment and show the movement of a segmentedflow of multiple fluid species through the system.

FIG. 2 provides a schematic diagram of a diffusion minimization systemaccording to one embodiment.

FIG. 3 provides a schematic diagram of a diffusion minimization systemaccording to one embodiment.

FIG. 4 provides a schematic diagram of a diffusion minimization systemaccording to one embodiment.

FIG. 5 provides a schematic diagram of system components that may beused in conjunction with a diffusion effect minimization systemaccording to one embodiment.

FIG. 6 is a flowchart illustrating a diffusion minimization processaccording to one embodiment.

FIG. 7 illustrates a functional block diagram of a microfluidic systemaccording to one embodiment.

FIG. 8 illustrates a top view of a portion of a microfluidic chipaccording to one embodiment.

FIGS. 9A-9D show the movement of a segmented flow of multiple fluidspecies through a diffusion minimization system according to oneembodiment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the systems and methods of creating and moving segmentedflows of multiple miscible fluid species through a microfluidic system,while minimizing diffusion effects between the fluid species, aredescribed herein with reference to figures. In the illustratedembodiments, regions of fluid that become diffused during theirtransport through the microfluidic system are diverted or vented awayfrom a collection or measurement area such that only regions of fluidwith no or little diffusion are sent to the collection or measurementarea.

FIGS. 1A-1D illustrate a diffusion minimization system 101 for theelimination and/or minimization of diffusion effects according to oneembodiment of the present invention. As shown in FIGS. 1A-D, diffusionminimization system 101 has pathways or channels A-C, which are in fluidcommunication with each other (i.e., fluid may flow from one of channelsA-C into another of channels A-C). In one embodiment, fluid enters thesystem via pathway A, and leaves the system via pathways B and C.Pathway B may lead to a microfluidic channel where detection,measurement and/or collection may be carried out, and where pure orsubstantially pure fluid is desirable. Pathway C may lead to a wastearea of a microfluidic system. In one embodiment, the fluid entering thesystem via pathway A may be from one or more test solution reservoirs.

As illustrated in FIG. 1A, in accordance with one embodiment, allpathways of the system 101 are primed with a first species of fluid(shown with horizontal hatch marks). Then a second species of fluid(shown in vertical hatch marks) is introduced to the system via pathwayA. A diffusion zone (shown with cross hatches) comprises a portion ofthe fluid flow that is a mixture of the first and second species offluid and will exist between a pure portion of the first fluid and apure portion of the second fluid. As explained above, the extent towhich the diffusion occurs may be exacerbated by the parabolic velocityprofile of microfluidic flow. In one embodiment, as the diffusion zonenears the entrance to pathway B, the flow into pathway B is shut off toprevent mixed, non-pure fluid from flowing into pathway B and into, forexample, the microfluidic channel. The entire diffusion zone is thenpumped out through pathway C, as illustrated in FIG. 1B. Once thediffusion zone has passed the entrance to pathway B, pathway B is openedagain and the pure second species of fluid is allowed to flow intopathway B, as illustrated in FIG. 1C.

This cycle of accepting regions of pure fluid and venting or divertingregions of diffused fluids may be a repeatable process. For example,FIG. 1C shows another diffusion zone of between a pure portion of secondfluid and a pure portion of the next liquid (shown in horizontal hatchmarks). As the diffusion zone approaches pathway B, as shown in FIG. 1D,the flow into pathway B is again shut off. As described above, theentire diffusion zone is then pumped out through pathway C, and, oncethe diffusion zone has passed the entrance to pathway B, pathway B isopened again and the pure fluid is allowed to flow into pathway B. Thisallows miscible fluids to be transported through a system in segmentedform because the contaminated, non-pure diffusion zones between thefluid segments are vented away or discarded before the pure fluids entera microfluidic channel for detection, measurement and/or collection.Thus, the diffusion minimization system 101 may also be thought of as azero dead volume connection between two parts. By placing the connectionbetween the parts where pathway C branches off from pathway B, any deadvolume may be eliminated through the repeated flushing of contaminatedfluid.

One or more diffusion minimization systems 101 may be located in adiffusion minimization or elimination zone of a microfluidic systemhaving one or more microfluidic channels. An example of such amicrofluidic system is illustrated in FIG. 7, described in detail below.In one embodiment, one diffusion elimination system 101 is provided for,and is in fluid communication with, each microfluidic channel of amicrofluidic system.

Many other embodiments of the diffusion minimization system can also becreated. For example, FIGS. 2-4 show diffusion minimization systems 201,301 and 401, respectively, in accordance with alternative embodiments ofthe present invention. Similar to the embodiment shown in FIG. 1, in theembodiments shown in FIGS. 2-4, fluid enters the diffusion minimizationsystem via pathway A, portions of fluids of a pure species are pulledinto pathway B, and any regions of fluid where diffusion has occurredare vented out of the system through pathway C. As discussed above, thepure species pulled into pathway B may lead to a microfluidic channel ina microfluidic system where detection, measurement and/or collection maybe carried out. In addition, an embodiment of the present invention mayinclude alternate configurations of a diffusion minimization systemhaving the same or similar characteristics and results of systems 101,201, 301 and 401.

Diffusion minimization system 401, as shown in FIG. 4, has the addedbenefit that fluid is pulled into pathway B, which may lead to amicrofluidic channel, from the center of the flow that comes frompathway A. This feature is in contrast to other embodiments which maypreferentially pull fluid from the edge of the pathway. Due to the lowervelocities associated with the edge of the flow, the fluid at the edgesof the flow will be the most diffused and will take the longest to flushclear. Accordingly, pulling fluid from the center of the pathway A intopathway B, as illustrated in FIG. 4, advantageously avoids the diffusedboundary layer at the edges of the flow.

The diffusion minimization system embodiments described above share thesame premise of preferentially pulling non-diffused regions of fluid offa channel that contains segmented regions of different fluid species,and then venting the diffused regions of flow off to a waste channel.This allows the collection of fluid that is not contaminated viadiffusion. The diffusion minimization system embodiments also allow formeasurements, which are functions of fluid concentration, to occur fromfluid in a single channel which contains multiple miscible flows stackedaxially in the channel. As other embodiments of the diffusionminimization system of the present invention may be imagined by makingvariations on the above geometries, the present invention is not limitedto any of the particular embodiments of the diffusion minimizationsystems described above. Indeed, an embodiment of the present inventionmay include alternate configurations of a diffusion minimization systemhaving the same or similar characteristics and results of systems 101,201, 301 and 401.

FIG. 5 shows an example of system components that may be utilized inconjunction with a diffusion minimization system. The diffusionminimization system 501 shown in FIG. 5 represents a diffusionminimization system, such as any of the embodiments described above. Forexample, diffusion minimization system 501 may be any of systems 101,201, 301 and 401 of FIGS. 1-4, respectively, or may comprise alternateconfigurations having the same or similar characteristics and results ofsystems 101, 201, 301 and 401. Like the diffusion minimization systemembodiments described above, system 501 may have channels A-C or someother arrangement of fluid flow channels that allows for selectivepassage of only pure fluid species segments into, for example, amicrofluidic channel while diverting away from the microfluidic channelmixed, non-pure fluids of a diffusion zone between adjacent segments ofdifferent fluid species.

As illustrated in FIG. 5, fluid entering the system 501 via pathway Amay be from test solution reservoir 550. Fluid flow in each of thechannels A-C may be controlled by fluid control system 519. Fluidcontrol system 519 may be configured to provide individual fluid controlmechanisms for each of channels A-C. For instance, the individual fluidcontrol mechanisms of fluid control system 519 may be present at theends of both channels B and C of system 501. Fluid control system 519may employ any mechanism of fluid control known in the art, such asvacuum pressure, positive pressure, and electrokinetics, to move thefluid species through the diffusion minimization system 501. In someembodiments, fluid control may also be provided by a fluid controlmechanism (e.g. vacuum pressure, positive pressure, and electrokinetics)acting on the microfluidic channel through which a pure fluid samplefrom channel B, for example, would be introduced.

In addition, as illustrated in FIG. 5, one or more monitoring devices520 may be used to identify and track the movement of the diffusionregions. In some embodiments, fluid control system 519 may useinformation from the monitoring device 520 to control the flow of thefluids through the channels in the diffusion minimization system. Inother embodiments, the monitoring device may comprise a video system toidentify and track the movement of the diffusion regions. Although fluidcontrol system 519 and monitoring device 520 are shown in FIG. 5 asbeing separate from diffusion minimization system 501, fluid controlsystem 519 and monitoring device 520 may be considered part of diffusionminimization system 501. In other embodiments, direct monitoring of thediffusion region is not required if the fluid velocity is known. Forexample, a set pressure or flowrate may be used to pull the segmentedflow of miscible fluids through the channels for a predetermined amountof time which is known to be sufficient to ensure that the diffusedregions would be past the collection point. This would allow that thenext fluid that is collected into pathway B, for example, issubstantially free of contamination from diffusion from other misciblefluids. Similarly, the collection of the non-diffused fluid into pathwayB can be stopped prior to the next diffusion region reaching pathway Bif the fluid velocity is known. In other embodiments, a fixed volumetricdisplacement could be used in place of or in addition to flowing thefluid at a given flow rate for a set amount of time. In one example,fluid control system 519 may be configured to use the fluid velocityand/or volumetric displacement to control the flowing, collecting andstopping of the fluids through the channels in the diffusionminimization system 501.

Referring now to FIG. 6, a flow chart is provided that illustrates aprocess of minimizing or eliminating diffusion effects in the segmentedflow of multiple, miscible fluid species in accordance with oneembodiment of the invention. The process shown in FIG. 6 may be carriedout, for example, with any of the diffusion minimization systems 101,201, 301 or 401 shown in FIGS. 1-4, respectively, or may be carried outwith alternate configurations having the same or similar characteristicsand results of systems 101, 201, 301 and 401.

In step 601, a first fluid species of a segmented flow of multiple,miscible fluid species is introduced into a first channel (e.g., pathwayA of FIGS. 1-4) of a diffusion minimization system. The fluid species ofthe segmented flow of multiple, miscible fluid species may be providedto the first channel from, for example, test solution reservoir 550. Instep 602, the first miscible fluid species (which may be free ofcontamination) is collected in a second channel (e.g., pathway B ofFIGS. 1-4) of the diffusion minimization system. In one embodiment, thesecond channel may lead to a microfluidic channel for PCR processing andHRTm analysis. In step 603, a second fluid species of the segmented flowof multiple, miscible fluid species is introduced into the firstchannel. In step 604, the diffusion region between the first and secondmiscible fluid species is identified, in some exemplary embodiments, viamonitoring or via knowledge of other parameters, including a combinationof the fluid velocity and the elapsed time, for example. In embodimentswhere the diffusion region is monitored, the monitoring of the diffusionregion may be performed by monitoring device 520. Provided that thediffusion region is not near the entrance to the second channel, thefirst miscible fluid species may continue to be collected in the secondchannel.

In step 605, when a diffusion region identified in step 604 isdetermined to be approaching the entrance to the second channel, forexample, via monitoring or via knowledge of other parameters (e.g.,fluid velocity and/or volumetric displacement), flow of fluid into thesecond channel is prevented. For example, a diffusion region may bedetermined to be approaching the entrance to the second channel by usingmonitoring device 520, or by using the fluid velocity in combinationwith the elapsed time, to determine that the leading edge of thediffusion region is within a predetermined distance from the entrance tothe second channel. The flow of fluid containing a diffusion region isthen diverted into a third channel (e.g., pathway C of FIGS. 1-4), whichmay be connected to a waste channel or reservoir, instead of beingpermitted to flow into the second channel.

In step 606, after the diffusion region has been determined to havepassed far enough into the third channel and away from the entrance tothe second channel, flow of fluid into the second channel is enabled,and the non-diffused portion of the second fluid species is collected inthe second channel. For example, a diffusion region may be determined tohave passed far enough into the third channel when the monitoring device520 determines that the trailing edge of the diffusion region is morethan a predetermined distance from the entrance to the second channel,or when it is determined that a specific amount of time has elapsed. Thesecond fluid species may be collected in the second channel undercontrol of fluid control system 519. In this way, the third channel actsas a vent through which the contaminated diffusion regions may beremoved from the segmented flow of multiple, miscible fluid species sothat only uncontaminated portions fluid species are provided formeasurement and detection.

In step 607, when a third fluid species of the segmented flow ofmultiple, miscible fluid species is introduced into the first channel,the steps 604-607 are repeated to vent contaminated, diffusion regionsbetween the adjacent second and third fluid species in the segmentedflow and to allow only non-diffused portions of the third fluid speciesinto the second channel.

The process described in connection with the embodiment shown in FIG. 6begins with introducing a first miscible fluid into the first channeland collecting that first fluid (which would be substantially free ofcontamination because it is not diffusing into another fluid) into thesecond channel which may lead to a microfluidic channel in amicrofluidic system where detection, measurement and/or collection maybe carried out. However, in other embodiments, a flush fluid may befirst introduced into all channels followed by the introduction of asecond miscible fluid. In this embodiment, the diffusion regions betweenthe first flush fluid and the second fluid may be vented into the thirdchannel and the second fluid, substantially free of contamination, wouldbe collected in the second channel. In still other embodiments, theprocess could involve identifying and monitoring the position of anydiffusion region between uncontaminated portions of adjacent misciblefluids, diverting the diffusion region into the second channel, andcollecting the portion of the uncontaminated fluid, in either one orboth of the adjacent miscible fluids, for further detection, measurementand/or collection. And, in other embodiments, the diffusion region isnot directly monitored, and the fluid velocity and/or fixed volumetricdisplacement is used determine whether to divert or collect the fluid.For example, a set pressure or flowrate may be used to pull thesegmented flow of miscible fluids through the channels for apredetermined amount of time and/or for a predetermined displacementvolume which is known to be sufficient to ensure that the diffusedregions would be past the collection point. This would allow the nextfluid that is collected into the second channel to be substantially freeof contamination from diffusion from other miscible fluids. Similarly,the collection of the non-diffused fluid into the second channel can bestopped prior to the next diffusion region reaching the second channelif the fluid velocity and/or displacement volume is known.

FIG. 7 illustrates an example of a microfluidic system 700, according toone embodiment of the invention, in which any of the diffusionminimization systems described above may be incorporated. As shown inFIG. 7, microfluidic system 700 may include a diffusion minimizationsystem 501, a microfluidic chip 702 having a PCR processing zone 704(i.e., a zone in which DNA is amplified) and a HRTm analysis zone 706(i.e., a zone in which the dissociation behavior of the amplified DNA isexamined).

FIG. 8 is a top view of microfluidic chip 702. As shown in FIG. 8,microfluidic chip 702 may include a number of microfluidic channels 802a-d. In the example shown, there are 4 microfluidic channels, but it iscontemplated that chip 702 may have more or less than 4 channels. Asshown, a first portion of each microfluidic channel may be within thePCR processing zone 704 and a second portion of each microfluidicchannel may be within the HRTm analysis zone 706.

When microfluidic system 700 is in use, at least one channel 802receives a sample (or “bolus”) of a solution (or “fluid”) containingreal-time PCR reagents from, for example, diffusion minimization system501. A force may be used to cause the bolus to travel through thechannel such that the bolus traverses PCR zone 704 prior to enteringHRTm zone 706. One system and method for performing PCR in amicrofluidic device is disclosed in U.S. patent application Ser. No.11/606,006, filed on Nov. 30, 2006 (U.S. Patent Application PublicationNo. 2008-0003593), incorporated herein by reference in its entirety.

The microfluidic system 700 may further include an image sensor 708, acontroller 710 for controlling image sensor 708, and an image processingsystem 712 for processing the image data produced by image sensor 708.Image sensor 708 may have a first image sensor region 721 and a secondimage sensor region 722. Image sensor 708 may be positioned with respectto microfluidic chip 702 such that at least a portion of PCR processingzone 704 is within the field of view of sensor region 721 and at least aportion of HRTm zone 706 is within the field of view of sensor region722.

Image sensor 708 may be used to (i) produce data corresponding to theintensity of emissions from PCR zone 704, and (ii) produce datacorresponding to the intensity of emissions from HRTm zone 706. Thus,microfluidic system 700 may simultaneously monitor (1) the amplificationof a sample of DNA, and (2) the dissociation behavior of a different DNAsample.

As illustrated in FIG. 7, microfluidic system 700 may include one ormore thermal generating apparatuses. In the embodiment shown,microfluidic system 700 includes a first thermal generating apparatus714 and a second thermal generating apparatus 716 and a controller 718for controlling apparatuses 714, 716. Each thermal generating apparatus714, 716 may be configured to provide heat to and/or absorb heat fromchip 702, and, thus, may include one or more heat sources and/or heatsinks. In some embodiments, the first thermal generating apparatus 714is configured such that while a bolus is within zone 704, thermalgenerating apparatus 714 cycles the temperature in zone 704 to achievePCR, and thermal generating apparatus 716 is configured such that, whena bolus enters zone 706, thermal generating apparatus 716 provides asubstantially steadily increasing amount of heat to zone 706 to causethe bolus to undergo HRTm analysis (i.e., to cause the dsDNA in thebolus to transition to ssDNA).

In some embodiments, microfluidic system 700 may further include anexcitation source 730 (e.g., a laser or other excitation source) forilluminating zones 704 and/or 706. Additional excitation sources (e.g.,source 731) may also be employed. Microfluidic system 700 may furtherinclude a lens 740 that is disposed between chip 702 and image sensor708. In such embodiments, lens 740 may be configured to focus onto thefirst image sensor region 721 light 745 coming from the PCR processingzone 704, and to focus onto the second image sensor region 722 light 746coming from the HRTm analysis zone 706.

In one embodiment, the diffusion minimization system 501, fluid controlsystem 519 and monitoring device 520 may operate to supply pure orsubstantially pure fluids to one or more microfluidic channels 802substantially as described above in connection with FIGS. 1-6. Althoughmicrofluidic system 700 is shown with a monitoring device 520, it is notnecessary that microfluidic system 700 include a monitoring device. Asdescribed herein, a known fluid velocity and/or volumetric displacementmay be used instead of direct monitoring of diffusion regions. In FIG.7, diffusion effect minimization or elimination system 501 is shown asbeing a component of microfluidic chip 702. However, in otherembodiments, diffusion minimization system 501 may be separate from, butfluidically connected to, microfluidic chip 702.

FIGS. 9A-9D show movement of segmented flows of multiple miscible fluidspecies through an exemplary diffusion effect minimization orelimination system in accordance with an embodiment of the presentinvention. FIGS. 9A-D show a first channel 901, a second channel 902 anda collection region 903. In the illustrated embodiment, collectionregion 903 is located at a junction formed between the first channel 901and the second channel 902. Similar to the embodiments shown in FIGS.1-4, in the embodiment shown in FIG. 9, fluid enters the diffusionminimization system via first channel 901, portions of fluids of a purespecies are pulled into second channel 902, and any regions of fluidwhere diffusion has occurred are vented out of the system through theportion of first channel 901 past the collection region 903. Asdiscussed above, the pure species pulled into the second channel 902 maylead to a microfluidic channel, such as a PCR channel, in a microfluidicsystem where detection, measurement and/or collection may be carriedout.

In FIGS. 9A-D, the white regions and the dark regions representdifferent miscible fluids flowing through first and second channels 901and 902. As illustrated in first channel 901, a diffusion region(represented in gray) exists between adjacent miscible fluidsrepresented by the white and dark segments.

As illustrated in FIG. 9A, the white and dark fluid segments flow from asupply to the first channel 901. As shown in FIG. 9A, the white fluidsegment has been prevented from entering and being collected in thesecond channel 902 leading, for example, to a PCR channel. The diffusionregion between the white fluid segment and the preceding dark fluidsegment has passed the collection region 903 and as moved towards awaste area. As shown, the diffusion region between the white fluidsegment and the subsequent dark fluid segment is a distance away fromthe collection region 903.

In FIG. 9B, the white fluid segment at the collection region 903 iscollected in the second channel 902 leading, for example, to the PCRchannel. The white fluid segment collected in the second channel 902 issubstantially free from contamination because the diffusion regionsbetween the adjacent dark fluid segments are away from the collectionzone. As the diffusion region between the white fluid segment and thesubsequent dark fluid segment approaches the collection region 903,fluid is again prevented from entering and being collected in the secondchannel 902. Accordingly, as shown in FIG. 9C, the diffusion regionspasses the collection region 903 without being collected, and flowsalong the first channel 901 towards the waste area. In this way, thecollected fluid samples in the second channel 902 remain substantiallyfree from of contamination of the diffusion region.

As shown in FIG. 9D, when the diffusion regions between the dark andwhite fluid segments are away from the collection region 903, the darkfluid segment, which is substantially free from contamination, iscollected in the second channel 902, which leads, for example, to thePCR channel. FIGS. 9A-D illustrate that alternating segments of misciblefluids that are substantially free from contamination may be collectedand flow into the second channel 902, as illustrated by the sharp whiteand dark boundaries between segments in that channel.

The embodiment illustrated in FIGS. 9A-D further includes a fluidcontrol system for controlling fluid movement in the channels and thecollection of the pure or substantially pure portion of the fluidsamples. In some embodiments, the fluid flow control system isconfigured to flow a continuous flow of the two or more miscible fluidsthrough the first channel 901 for a predetermined time, wherein thepredetermined time is sufficient to allow the diffused region of the atleast two miscible fluids to pass the collection region 903, to collecta portion of one of the miscible fluids, which is substantially freefrom contamination by any miscible fluids adjacent to the collectedmiscible fluid, into the second channel 902, and to stop the collectionof one of the miscible fluids before a subsequent diffused regionreaches the collection region. In some embodiments, the fluid flowcontrol system is configured to determine the predetermined time byusing a predetermined flow rate and/or a fixed volume displacement, asdiscussed herein. The fluid flow control system is also configured torepeat the flowing, collecting and stopping for each sample of misciblefluid which is substantially free from contamination by the othermiscible fluids to be collected.

Although the diffusion minimization system shown in FIGS. 9A-9B is shownwith a T-junction, the diffusion minimization system may comprise theone of the systems 101, 201, 301 and 401 of FIGS. 1-4, respectively, ormay comprise alternate configurations having the same or similarcharacteristics and results of systems 101, 201, 301 and 401. Amonitoring device 520 may be used to directly monitor the movement ofthe diffusion regions through the system, or the fluid velocity and/orvolumetric displacement may be used to control flow of the fluidsthrough channels for a time sufficient to ensure that the diffusedregions would be past the collection point, as discussed herein.

While the invention has been particularly taught and described withreference to certain embodiments, those versed in the art willappreciate that modifications in form and detail may be made withoutdeparting from the spirit and scope of the invention. For example, theflow in the first microfluidic pathway A may be continuous or may bestopped while a given miscible fluid mixture flows into the secondmicrofluidic pathway. Also, the flow into the second microfluidicpathway may be stopped or may flow in reverse while the diffused regionbetween the original two microfluidic segments in the first channel isallowed to pass by the entrance to the second channel. In addition, eachmiscible fluid mixture, as referenced above, may comprise only onefluid, a homogenous mixture of multiple fluids or a heterogeneousmixture of multiple fluids. Further, the processes herein described maybe repeated consecutively for multiple varied miscible fluid mixtures inthe practice of the present invention.

Moreover, although embodiments of the system and method for minimizingor eliminating diffusion effects have been described in relation to amicrofluidic system and method for nucleic acid analysis, the system andmethod of the present invention may be used to minimize or eliminatediffusion effects in any type of microfluidic system. And, althoughembodiments of the system and method for minimizing or eliminatingdiffusion effects have been described with the multiple, miscible fluidspecies collected in the second channel as being axially adjacent toeach other, the fluid control system may provide gaps between themultiple fluid species collected in the second channel after thediffusion regions have been flushed.

The invention claimed is:
 1. A method of collecting, from a continuousflow of two or more miscible fluids sequentially present in a channel,one or more samples that are substantially free from contamination bythe other miscible fluids present in the channel, said methodcomprising: a. identifying and monitoring the position of a diffusionregion between uncontaminated portions of a first miscible fluid and asecond miscible fluid in a first channel; b. diverting the diffusionregion into a second channel; and c. collecting a portion of the secondmiscible fluid which is substantially free from contamination by anymiscible fluids adjacent to the second miscible fluid.
 2. The method ofclaim 1, wherein the monitoring the position of the diffusion regionincludes monitoring a leading edge and a trailing edge of the diffusionregion.
 3. The method of claim 1, wherein the portion of the secondmiscible fluid is collected in a third channel.
 4. The method of claim3, wherein the position of the diffusion region is monitored in relationto an opening of the third channel.
 5. The method of claim 3, whereinthe diverting begins at or before a leading edge of the diffusion regionreaches an opening of the third channel and ends after a trailing edgeof the diffusion region passes the opening of the third channel.
 6. Themethod of claim 3, wherein the second channel leads to a waste area. 7.The method of claim 1, wherein the diverting pulls fluid of thediffusion region from the edge of the first channel and the collectingpulls fluid of the portion of the second miscible fluid from the centerof the first channel.
 8. The method of claim 1, further comprising: d.identifying and monitoring the position of a diffusion region betweenuncontaminated portions of the second miscible fluid and a thirdmiscible fluid in a first channel; e. diverting the diffusion regioninto the second channel; and f. collecting a portion of the thirdmiscible fluid which is substantially free from contamination by anymiscible fluids adjacent to the third miscible fluid.
 9. The method ofclaim 1, further comprising: d. monitoring the amplification of DNA inthe collected portion of the second miscible fluid; and e. monitoringthe dissociation behavior of the amplified DNA in the collected portionof the second miscible fluid.
 10. A genomic analysis system comprising:a microfluidic chip having: one or more microfluidic channels eachpassing through a diffusion minimization zone, a polymerase chainreaction processing zone, and a high-resolution thermal melt analysiszone; and a diffusion minimization system in fluid communication withone or more microfluidic channels, wherein the diffusion minimizationsystem comprises: a first pathway through which a segmented flow ofmultiple, miscible fluid species enters; a second pathway through whichone or more miscible fluid samples that are substantially free fromcontamination by adjacent miscible fluids present in the first pathwayare collected and passed to the PCR processing zone and the HRTmanalysis zone; and a third pathway through which one or more diffusionregions between adjacent fluid species of the segmented flow arediverted to a waste zone; a monitoring device that identifies and tracksthe one or more diffusion regions present in the diffusion minimizationzone of the microfluidic chip; and a fluid flow control systemconfigured to control the flow of fluid through the diffusionminimization system and through the one or more microfluidic channels.11. A method of collecting a sample from a continuous flow of two ormore miscible fluids sequentially present in a channel, comprising:identifying the leading region of diffusion between two sequentialfluids, monitoring the passage of the region of diffusion through thechannel such that once the leading region of diffusion has passed anopening for a second channel a portion of the fluid is collected into asecond channel, wherein the collecting of fluid is stopped before thenext sequential region of diffusion reaches the opening of the secondchannel.
 12. The method of claim 11 wherein the collected sample is freefrom contamination by the other miscible fluid(s) present in thechannel.
 13. A method of collecting, from a continuous flow of two ormore miscible fluids sequentially present in a first channel, one ormore samples that are substantially free from contamination by the othermiscible fluids present in the first channel, said method comprising: a.flowing the continuous flow of two or more miscible fluids through thefirst channel for a predetermined time, wherein the predetermined timeis sufficient to allow the diffused region of the at least two misciblefluids to pass a collection region of the first channel, wherein thecollection region of the first channel is a junction with a secondchannel; b. collecting a portion of one of the miscible fluids, which issubstantially free from contamination by any miscible fluids adjacent tothe collected miscible fluid, into the second channel; and c. stoppingthe collection of one of the miscible fluids before a subsequentdiffused region reaches the collection region of the first channel. 14.The method of claim 13, wherein the predetermined time is determined byusing a predetermined flow rate and/or a fixed volume displacement. 15.The method of claim 13, wherein steps a-c are repeated for each sampleof miscible fluid which is substantially free from contamination by theother miscible fluids to be collected.
 16. The method of claim 13,wherein the either one or both of the first and second channels is amicrofluidic channel.